Lung cancer is a major research priority for the Department of Veterans Affairs. In the United States and among our veteran population lung cancer remains the leading cause of cancer death. Despite the existing therapeutic efforts, the long term survival for lung cancer patients remains low, thus new therapeutic strategies are needed. In the proposed study we will evaluate an immune based therapy for lung cancer utilizing an orthotopic murine tumor model. We will determine the immune and innate effectors required in the generation of secondary lymphoid chemokine gene modified stromal cells (CCL21-SC) mediated antitumor activity and the role of regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC) on the efficacy of CCL21-SC therapy. Our overall hypothesis is that CCL21-SC therapy will modulate antitumor responses in lung cancer. Hypothesis 1: Depleting CXCR3 and CCR7 receptor expressing effector cells [immune (CD4 T and CD8 T) and innate (NK and NKT)] will reduce CCL21-SC mediated antitumor activity. Hypothesis 2: Decreasing Treg and/or MDSC activity will improve the efficacy of CCL21-SC therapy. Specific Aims: Generation of antitumor effectors is a critical factor for successful immune based therapies. In aim 1, we will define the cellular requirements for the generation of CCL21-SC mediated antitumor responses in vivo by individually neutralizing CXCR3, CCR7, CD4, CD8, and NK cells with antibodies. The role of NKT cells will be defined utilizing the NKT deficient CD1d -/- mouse. The effect of specific effector cell neutralization on CCL21-SC therapy will be monitored by quantifying changes in: tumor burden, survival, the frequencies and functional activities of immune (CD4 T and CD8 T) and innate effector cells (NK and NKT). An understanding of the requisite effectors in the generation of CCL21-SC mediated antitumor responses will enable us to monitor similar cellular biomarkers in a lung cancer clinical trial of this therapy. Our preliminary findings indicate that the CCL21-SC therapy is very effective against tumor development in the subcutaneous (5-day) and orthotopic (7-day) Lewis Lung (3LL) tumor models. However, when therapy is initiated at a later stage of tumor development (subcutaneous 10-day or orthotopic 21-day), the efficacy of therapy is attenuated. The tumor uses strategies to counteract the immune responses that seek to destroy it. Tumor immune suppression can occur through the induction and recruitment of Treg and MDSC that block activation of immune and innate effectors. Whereas the frequency of Treg and MDSC is low in early stage tumor, they increase in later stage tumors. Thus, it is possible that Treg and/or MDSC are attenuating CCL21-SC therapy by reducing the activation and expansion of T lymphocytes and innate effector cells. In aim 2, to define the role of Treg or MDSC on the efficacy of CCL21-SC therapy, we will individually inactivate Treg or neutralize MDSC suppressor cell activity with antibodies. The impact of Treg inactivation or MDSC neutralization on the efficacy of CCL21-SC therapy will be monitored by quantifying changes in tumor burden and survival that will be correlated with changes in the frequencies and functional activities of immune and innate effectors. The impact of Treg inactivation or MDSC neutralization on CCL21-SC therapy will provide evidence for targeting of these suppressor phenotypes for enhancing CCL21-SC mediated antitumor activity. Findings from this study can lead to the development of innovative and effective immune therapies for lung cancer.